Jak2 Phosphorylates Tyr 177 of Bcr-Abl Activating the Ras and PI-3 Kinase Pathways and Maintains Functional Levels of Bcr-Abl in Chronic Myelogenous Leukemia.

Abstract

Abstract 39We have shown that Jak2 is activated in Bcr-Abl+ hematopoietic cells. Our findings indicate that Jak2 kinase inhibition overcomes drug-resistance in CML cells (Samanta et al., 2006). Recently, we showed that Jak2 inhibition deactivates the Lyn kinase in Bcr-Abl+ cells by inhibiting SET expression; SET inhibits the PP2A-SHP1 (Samanta et al., 2009). Because of the critical effects of Jak2 in Bcr-Abl+ cells, we explored the mechanism of Jak2 action in CML cells. Bcr-Abl is known to be phosphorylated on a number of tyrosine residues including tyrosine 177. Phosphorylation of Tyr 177 leads to binding to Grb2, activation of the SOS-Ras pathway, and activation of the PI-3 kinase through the Grb2-Gab2 pathway. Here, we show that Jak2 and not Bcr-Abl phosphorylates Tyr 177 of Bcr-Abl. Tyr 177 of Bcr-Abl (YVNV) has the Jak2 consensus target sequence (YxxV/L/I) (Argetsinger et al., 2004) as do a number of other Tyr residues in the Bcr portion of Bcr-Abl (b3a2). Inhibition of Jak2 by a specific Jak2 kinase inhibitor TG101209 (TargeGen Inc., San Diego, CA) and a new Jak2 kinase inhibitor (WP1193, synthesized by Dr. Priebe) in Bcr-Abl+ cells, but not inhibition of Bcr-Abl by imatinib mesylate (IM), rapidly reduced phosphorylation of Tyr 177 of Bcr-Abl in IM-sensitive and –resistant cell lines including K562, K562-R, Bcr-Abl+ 32D, Bcr-Abl+ BaF3, Bcr-Abl+T315I BaF3 cells and cells from blast crisis CML patients. Jak2 inhibitors but not IM also strongly inhibited the phosphorylation of a synthetic Bcr peptide containing Tyr 177 (and the surrounding Bcr sequences) catalyzed by either recombinant purified Jak2 or Jak2 immune complexes. Jak2 inhibition of Bcr-Abl+ 32D cells rapidly reduced Grb2 binding to Bcr-Abl, and diminished activation of Ras and PI-3 kinase pathways within two h. Importantly, knockdown of Jak2 with Jak2-specific siRNA also reduced levels of pTyr 177 Bcr-Abl and total Bcr-Abl protein. Rescue experiments reversed Jak2 knockdown and stimulated pTyr 177 levels. In whole cells the total levels of the Bcr-Abl protein also decreased upon Jak2 inhibition with the above chemical inhibitors but at a rate slower than the rate observed in the inhibition of the Tyr 177 phosphorylation of Bcr-Abl. In kinase assays with Bcr-Abl network complexes from Bcr-Abl+ cells, phosphorylation of Tyr 177 of Bcr-Abl was drastically reduced by Jak2 inhibition but Bcr-Abl protein levels remained stable. Reduction of Bcr-Abl protein levels in Bcr-Abl+ cells by Jak2 inhibition also caused a severe reduction of tyrosine phosphorylation of STAT5 and STAT3. We have shown by gel filtration column chromatography of lysates isolated from Bcr-Abl+ cells that Bcr-Abl is physically associated with the IL-3 receptor beta chain, Jak2, Akt and HSP90 in high molecular weight complexes. GSK3β, STAT5 and STAT3 also emerge from the column in the high molecular weight fractions and suggested that several high molecular weight complexes may exist. Nevertheless, incubation of Bcr-Abl+ cells with WP1193 caused disruption of these large molecular network complexes within three h. Nude mice, injected subcutaneously with IM-resistant K562-R cells developed solid tumors, which were reduced in weight by i.p. injection of WP1193 every 48 h at 30 mg/Kg over 10 days. WP1193 at a similar dosage strongly reduced leukemic and tumor effects of Bcr-Abl+ 32D cells expressing T315I Bcr-Abl injected i.v. into nude mice. WP1193 injections into nude mice did not cause significant toxicity over a 10 day period. In summary, Jak2 inhibitors TG101209/WP1193 rapidly reduced levels of pTyr 177 Bcr-Abl, reduced levels of Bcr-Abl protein and induced high levels of apoptosis in IM-sensitive and –resistant Bcr-Abl+ cell lines including T315I cells, CML cell lines, cells from CML patients in blast crisis. In addition Jak2 inhibition reduced tumor formation in mice injected with IM-resistant K562-R and T315I Bcr-Abl+ mouse cells. These findings establish: 1) Jak2 as a major driver of Bcr-Abl signaling pathways in Bcr-Abl+ CML cells, which were previously thought to be activated directly by Bcr-Abl. 2) Although Jak2 is activated by Bcr-Abl, Jak2 is the dominant target molecule in CML and Jak2 becomes even more dominant in CML cells that become resistant to IM and other tyrosine kinase inhibitors. These findings indicate that CML cells, because of the dominant role of Jak2, resemble leukemia cells from Bcr-Abl negative myeloproliferative diseases. Disclosures:No relevant conflicts of interest to declare.